Ophiostomatoid Fungi Associated with Cryphalus Piceae in Shandong province in eastern China

Cryphalus piceae parasitizes various economically important conifers. Similar to other bark beetles, C. picea vectors an assortment of fungi and nematodes. Previously, several ophiostomatoid fungi were isolated from C. piceae in Poland and Japan. In the present study, we explored the diversity of ophiostomatoid fungi associated with C. piceae infesting pines in the Shandong Province of China. We isolated ophiostomatoid fungi from both galleries and beetles collected from our study sites. These fungal isolates were identied using both molecular and morphological data. Through this study, we recovered 176 isolates of ophiostomatoid fungi representing at least seven species. Ophiostoma ips was the most frequently isolated species. Analyses of molecular and morphological data indicated four of the ophiostomatoid fungal species recovered in this study were previously undescribed. Hereby, we described these species as Ceratocystiopsis yantaiensis sp. nov., C. weihaiensis sp. nov., Graphilbum translucens sp. nov. and Sporothrix villosa sp. nov. A majority of the ophiostomatoid fungi recovered in this study were novel species. This suggests that the forests in China harbour an assortment of undescribed ophiostomatoid fungi yet to be discovered.

Ophiostomatoid fungi often form a symbiotic association with bark and ambrosia beetles who assist in the dispersal of their inocula (Klepzig & Six, 2004). For example, Ceratocystiopsis ranaculosus and Entomocorticium sp. A remain associated with the mycangium of D. frontalis whereas Ophiostoma minus is carried phoretically on the exoskeleton (Hofstetter et al., 2015). Moreover, an ophiostomatoid fungus can symbiotically associate with multiple beetle species. Recently, six ophiostomatoid fungi were isolated from M. alternatus in China (Zhao et  In Europe and Asia, Cryphalus piceae parasitizes various species of Abies, Pinus, Picea and Larix (Jankowiak & Kolarik, 2010). This bark beetle predominantly affects stressed trees (Michalski & Mazur, 1999), but can also infect healthy ones (Justesen et al., 2020). Previously, several fungal species were isolated from C. piceae. This included an assortment of ophiostomatoid fungi and Geosmithia from Poland (Jankowiak & Kolarik, 2010;Jankowiak & Bilanski, 2018) and Japan (Ohtaka et al., 2002a;Ohtaka et al., 2002b). In the present study, we explored the diversity of ophiostomatoid fungi associated with C. piecea from the pine plantations located in the Shandong Province of China.
Isolation of fungi was done using the method suggested by Chang et al. (2019). Fungal mycelium and/or spore masses from C. piecea galleries were transferred onto 2 % malt extract agar (MEA) medium amended 0.05 % streptomycin. In cases where no mycelia were visible, galleries were incubated in moist chambers at 25°C in darkness for 4-6 weeks. Post incubation, conidia with spore masses emerging from these incubated galleries were transferred onto MEA amended with streptomycin. For isolating fungi from the beetles, adult C. piecea were crushed on the surface of MEA amended streptomycin. In order to purify the fungal isolates, hyphal tips from colonies were transferred onto fresh MEA plates.
All fungal isolates were submitted to the microbial culture collection of Shandong Normal University, Jinan, Shandong, China (SNM; for accession numbers see Table 1). Ex-holotypes culture of ophiostomatoid fungi described in this study were deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. Holotype specimens (dry culture) were deposited in the Herbarium Mycologicum, Academiae Sinicae (HMAS), Beijing, China. Table 1 Isolates of ophiostomatoid fungi obtained from Cryphalus piceae and used in this study.  All the isolates obtained in this study were initially grouped based on colony morphologies. For the purpose of preliminary identi cation, at least two representative isolates from each group were identi ed using the molecular technique. For the novel species described in the present study, all isolates were sequenced to con rm their identity.
The PrepMan ultra sample preparation reagent (Applied Biosystems, Foster City, CA) was used for extracting the total genomic DNA from ve-day-old cultures, following the manufacturer's protocols. The complete ITS region and partial β-tubulin (BT), elongation factor 1-α (EF), and calmodulin (CAL) genes were ampli ed using primers ITS1F/ITS 4 ( Each 25 µl PCR reaction included 12.5 µl 2 × Taq Master Mix (buffer, dNTPs and Taq; Vazyme Biotech Co., Ltd, China), 0.5 µl each of forward and reverse primers, 10.5 µl PCR grade water, and 1 µl of DNA template. PCR ampli cations were conducted with an initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 60 sec; annealing temperature was 55 ºC for 60 sec for all primers; 72°C for 1 min; and nal elongation at 72°C for 10 min.
All the PCR products were sequenced by the Sangon Biotech, Qingdao, Shandong Province, China. The resulting sequences were assembled using Geneious v. 7.1.4 (Biomatters, Auckland, New Zealand). The BLAST algorithm (Altschul et al. 1990) available through the NCBI GenBank was used for the preliminary identi cation of the sequences. All the sequences were submitted to GenBank and the accession numbers are listed in Table 1.

Phylogenetic analyses
For the purpose of phylogenetic analyses, separate datasets were prepared for all four gene regions (ITS, BT, EF and CAL). Each of these datasets included sequences generated in this study, and those that were retrieved from the GenBank (including the ex-type sequences). We recovered multiple isolates of the same species from O. ips and S. gossypina complex. Therefore, datasets for these two complexes included sequences from at least four representative isolates. The dataset was aligned using MAFFT v. 7 ( . Four MCMC chains were run from a random starting tree for ve million generations and trees were sampled every 100th generation. One-fourth of the sampled trees were discarded as burn-in and the remaining trees were used for constructing majority rule consensus trees. MEGA-X was used for conducting maximum parsimony (MP) analyses (Kumar et al., 2018) where gaps were treated as a fth character.
Growth and morphological studies A representative isolate for each new fungal species identi ed through phylogenetic analyses was selected for morphological study.
Isolates were initially sub-cultured on 2% MEA and incubated for seven days at 25°C in darkness. Thereafter, 5 mm agar plugs were placed at the centres of 90 mm Petri dishes and three replicate plates per isolate were incubated at 5, 10, 15, 20, 25, 30 and 35 ºC (± 0.5 ºC) in darkness. The colony diameter of each isolate was measured at an interval of two days up to the tenth day.
Microscopic structures of the ophiostomatoid fungi were measured and photographed using a Zeiss Axio Imager Z2 (CarlZeiss, Germany). Fifty measurements for each taxonomically informative structure were made, such as conidiophore and conidia.

Collection of beetles and isolation of fungi
In the present study, 176 isolates of ophiostomatoid fungi were recovered. Among these, 148 were isolated from galleries whereas 28 from beetles. Based on the collection sites, 16 isolates were recovered from Yantai, 63 from Qingdao and 97 from Weihai.
Species residing in Ceratocystiopsis were analyzed using ITS and BT gene regions. In the phylogenies of Ceratocystiopsis, four isolates of Ceratocystiopsis recovered in this study clustered into two distinct monophyletic clades (Fig. 1). Taxon 1 (two isolates) and Taxon 2 (two isolates) are sister species to C. manitobensis and C. minuta, respectively (Fig. 1).
Species residing in Graphilbum were analyzed using ITS, BT and EF gene regions. Phylogenetic analyses of six isolates clustered them into two distinct monophyletic clades (Fig. 2). Taxon 3 (four isolates) is closely related to Gr. puerense and Gr. acuminatum whereas Taxon 4 (two isolates) was Gr. crescericum.
The identity of the isolate residing in Graphium was con rmed using ITS and EF gene regions. Taxon 5 (one isolate) was identi ed as G. pseudormiticum (Fig. S1).
Species resided in O. ipx complex were analyzed using ITS and BT gene regions. In the ITS and BT trees, our isolates (Taxon 6, 141 isolates) formed monophyletic clades with O. ips (Fig. S2).
Isolates from the S. gossypina complex were analyzed using ITS, BT and CAL gene regions. Phylogenetic analyses showed those isolates (Taxon 7, 24 isolates) were closely related to two fungal isolates from China that were previously identi ed as S. cf. abietina (Fig. 3). Culture characteristics: Colonies light brown on MEA (Fig. 4a). Mycelia white, super cially growing on the agar. The optimal temperature for growth was 30-35°C, reaching 43.0 mm diam in 10 days. No growth observed at 5°C.

Distribution
Currently known from Yantai City in Shandong Province, China.

Note
Ceratocystiopsis yantaiensis is phylogenetically close to C. manitobensis but formed a distinct clade on both ITS and BT trees (Fig. 1). Two types of hyalorhinocladiella-like asexual state were also observed in C. manitobensis (Hausner et al., 2003). Conidia of C.
yantanensis and C. manitobensis are similar in morphology, but the former is smaller in size (Fig. 4b-e).

Diagnosis
Compared to other closely related species, C. weihaiensis produces smaller conidia. Culture characteristics: Colonies light brown on MEA (Fig. 5a). Mycelia white, submerged in the agar. The optimal temperature for growth is 30°C, reaching 46.0 mm diam in 10 days. Growth is slower at 35°C, 27 mm diam in 10 days.

Distribution
Currently known from Weihai City in Shandong Province, China.
Culture characteristics: Colonies light brown on MEA (Fig. 6a). Mycelia submerged in the agar. The optimal temperature for growth is 30°C, reaching 74.0 mm diam in 5 days. Growth slower at 35°C, 24 mm diam in 5 days. No growth was observed at 5°C.

Distribution
Currently known from Qingdao City and Weihai City in Shandong Province, China.

Discussions
In the present study, we collected Cryphalus piceae and their galleries from various pine plantations located near Qingdao, Weihai and Yantai cities, Shandong province of China. From these beetles and galleries, we recovered 176 isolates of ophiostomatoid fungi representing ve well-de ned genera. These genera were Ceratocystiopsis, Graphilbum, Graphium, Ophiostoma and Sporothrix. Analyses of molecular and morphological data indicated four of the ophiostomatoid fungal species recovered in this study were previously undescribed. Hereby, we described these species as C. yantaiensis, C. weihaiensis, Gr. translucens and S. villosa.
Among the seven ophiostomatoid species identi ed in this study, resolving the taxonomy for two isolates of Gr. crescericum was particularly challenging. Sequences for ITS, EF and CAL from the European isolates of this fungus are available, however, BT is missing (Jankowiak et al., 2020). Whereas in the present study, we successfully ampli ed the BT gene (along with ITS and EF), but even after repeated attempts we could not amplify the CAL gene. While comparing the EF gene region, our isolates of Gr. crescericum and those from Europe had at least seven base pairs difference. Similar interspeci c variation for the ITS, EF, BT and CAL genes were also reported from O. quercus and O. tsotsi (Chang et al., 2017;Taerum et al., 2018). Therefore, in the future, different sets of primers should be designed for amplifying BT and CAL gene regions for Graphilbum. This will allow us to demystify the taxonomy of this genus.
Ophiostoma ips was one of the most frequently isolated ophiostomatoid fungi in China and this study (Lu et  Owing to climate change, economic damages caused by these bark beetles and nematodes has exponentially increased in China (Li, 2013;Tang et al., 2021). This initiated studies focusing on the biology and controlling of these beetles . These studies simultaneously catalogued the diversity of symbiotic fungi associated with these beetles, in uencing fungal species discovery Zhao & Sun, 2017).
In this study, we recovered seven species of ophiostomatoid fungi including four previously undescribed species from the Shandong province of China. The previous study from this province, reported two new ophiostomatoid fungi associated with B. xylophilus and M. alternatus collected from two pine species (Wang et al., 2018). Thus far, more than 10 bark beetle species have been reported from this province (Bai, 1985;Zhu et al., 1991). Previous to this study, no attempts were made to isolate ophiostomatoid fungi from Shandong.
Therefore, in future, follow-up surveys and isolations from other bark beetle species from the province will likely allow the discovery of several novel ophiostomatoid fungi.